Peninsula Laboratories, Llc
PENINSULA LABORATORIES, LLC
A Member of the bachem group
GENERAL PROTOCOL
FOR
Peptide Radioimmunoassay (RIA)
CAUTION: INVESTIGATIONAL DEVICE
LIMITED BY FEDERAL LAW TO INVESTIGATIONAL USE
FOR RESEARCH USE ONLY
NOT FOR USE IN DIAGNOSTIC PROCEDURES
305 Old County Rd. San Carlos, CA 94070
TEL: (650) 592-5392 (800) 922-1516 FAX: (650) 595-4071
RIA KITS - BASIC NOTIONS AND FACTS
Our RIA kits are competitive immunoassays whose working principle can be diagrammed as follows:
A constant concentration of 125I-tracer (125I radiolabeled tracer) and varying concentrations of unlabeled standard or sample peptide compete for binding specifically to the antiserum. Immunocomplexes are precipitated by adding an excess of non-specific serum and secondary polyclonal antiserum followed by centrifugation. Unbound reagents are washed and the pellet is counted in a gamma counter.
The sequence of the standard peptide is shown on the datasheet. It is also in our catalog and on our web site (www.BACHEM.com) as the "antigen sequence" (note that large protein sequences are usually not shown).
The standard is used to make a standard curve in the range specified in the kit's datasheet. Standard curves are S-shaped (on a semi-log plot) but for a few kits they appear to be almost linear over the kit's range. The measuring range is the range of standard concentrations near the middle or near the IC50 of the standard curve. Typical standard curves for most kits are printed in our catalog but can also be obtained via e-mail (). Unknown sample concentrations are measured by comparing its cpm with the standard curve.
We include sufficient reagents for 125 RIA test tubes.
VARIATION · ACCURACY · EXTRACTION · CROSSREACTIVITY
The kit's IC50, or the shape of the standard curve, may show some variation but this will not affect the kit's accuracy in the measuring range. The kit accurately measures sample peptides if the following conditions are met.
A) The kit's antiserum must not cross-react appreciably with other factors present in the sample. Cross-reactivity tables are included with each kit. The user may wish to test the cross-reactivity with other peptides, some of which may be available from our catalog.
B) The sample peptides must be identical to the kit's standard. Ideally the kit's synthetic standard mimics the natural peptide perfectly. Sometimes, however, natural peptides exist as families of species related by a common or similar sequence. In addition, natural peptides may be enzymatically or spontaneously modified, may exist in complexes, and may assume alternative structural forms. In these cases the kit might not measure the exact concentration of a particular natural peptide species, but, it may still be used for relative average measurements.
C) Serum samples must be extracted. Factors present in serum can bind to kit components and affect the measurements. Thus, it is advisable to extract peptides from serum before measuring them. Extraction may not be as important if the sample is diluted before measuring or if it is in tissue culture medium, but, in general, one may expect low sensitivity or high background problems if extraction is not used. Some antigens can be measured directly in un-extracted serum. If so, a specially formulated extraction-free kit may be available in our catalog or as a custom product, which can be used without extraction for human or rat serum.
D) Both samples and standards must be measured under the same conditions.
TECHNICAL HELP and ORDERING INFORMATION
Please e-mail technical questions to
For additional kits or listings of our most current catalog products, please visit our web site at www.bachem.com or call us at:
Tel: (800) 922-1516 (for U.S. and Canada)
(650) 592-5392 (for International)
Fax: (650) 595-4071
SAFETY PRECAUTIONS
The 125I radioisotope produces 35.5 KeV gamma radiation. It has a half-life of 60.1 days, and a biological half-life of 120 to 138 days. The amount of exposure from a 1.5 mCi tracer is 0.3 mR/Hr at contact. The critical organ for uptake is the thyroid gland. Please use shielding and equipment designed for 125I or gamma radiation.
This radioactive material shall be received, acquired, possessed and used by licensed investigators in laboratories or hospitals for in vitro laboratory tests only. Its use shall not involve internal or external administration of the material and radiation therefrom to human beings or animals. Its receipt, acquisition, possession, use and transfer are subject to the regulations and license requirements of the U.S. Nuclear Regulatory Commission or of a State with which the Commission has entered into an agreement for the exercise of regulatory authority.
The user should designate an area for handling 125I and clearly label all containers. There should be no drinking, eating, or smoking where 125I is being handled. Use transfer pipettes, spill trays, and absorbent coverings to confine contamination. All users should wear disposable lab coats, gloves, and wrist guards as a secondary precaution. Regularly monitor and promptly decontaminate gloves and surfaces to maintain contamination control. Use a NaI (TI) detector or liquid scintillation counter to detect 125I. Conduct periodic thyroid counts to determine an internal dose. Isolate waste in sealed, labeled containers.
Establish surface contamination, air concentration, and thyroid burden action levels below maximum limits and investigate any causes that threaten these levels to be exceeded. Persons under 18 years old should not be permitted to handle radioactive material or enter radioactive areas.
Disposal - Radioactive waste should be disposed of in compliance with Federal, State, and Local Government regulations.
THIS PACKAGE CONFORMS TO THE CONDITIONS AND LIMITATIONS SPECIFIED IN TITLE 49 CFR173.421 FOR RADIOACTIVE MATERIAL.
Some of the kit components (including the RIA buffer) contain Sodium Azide. Please handle and dispose of according to established safety regulations.
GUARANTEE AND LIMITATION OF REMEDY
PENINSULA LABORATORIES, LLC makes no guarantee of any kind, expressed or implied, which extends beyond the description of the material in this kit, except that these materials and this kit will meet our specifications at the time of delivery. The customer’s remedy and PENINSULA LABORATORIES, LLC’s sole liability hereunder are limited at PENINSULA LABORATORIES, LLC’s option to refund the purchase price or the replacement of material that does not meet our specifications. By the acceptance of our products, the customer indemnifies and holds PENINSULA LABORATORIES, LLC harmless against, and assumes all liability for the consequences of its use or misuse by the customer, its employees or others.
TRACER DECAY
· Tracers should be used as soon as possible.
· The specific activity of the 125I-tracer will decay with a half-life of 60.1 days (for example in 1 month the tracer will lose 30% of its radioactivity).
MATERIALS PROVIDED AND STORAGE CONDITIONS
· RIA buffer 4x concentrate 50 ml (Dilute to 200 ml). Store at 4°C before and after dilution.
· Standard peptide lyophilized powder (for 1 ml RIA buffer). Store at 4°C before re-hydration and at -20°C after rehydration.
· Specific rabbit or guinea pig antiserum lyophilized powder (for 13 ml RIA buffer). Store at 4°C before re-hydration and at -20°C after rehydration.
· 125I-tracer: at least 1.5 mCi (on preparation day). Store at -20°C.
· Secondary polyclonal antiserum lyophilized powder (for 13 ml RIA buffer), goat anti rabbit (GARGG) or goat anti-guinea pig (GAGGG) antiserum depending on the kit. Store at 4°C before and after re-hydration.
· Normal serum lyophilized powder (for 13 ml RIA buffer). Normal rabbit or normal guinea pig serum depending on the kit. Store at 4°C before and after re-hydration.
· Data sheet
· Standard Diluent 8 ml of peptide-free stripped frozen serum (for extraction-free kits only). Store at -20°C.
MATERIALS NOT PROVIDED
· SEP-COLUMN containing 200 mg of C18 Cat No. Y-1000) (optional)
· Extraction kit Cat No S-5000 (for 50 samples with buffers) (optional)
· 12 x 75 mm polystyrene tubes
· Gamma counter for the above tubes
· Centrifuge for the above tubes
· Curve fitting software (optional)
· Various other standard laboratory items ...
ASSAY PROTOCOL
We recommend using the same basic 3-day RIA protocol shown on the following page for all our RIA kits. Depending on the kit, the details of the protocol differ with respect to three parameters: extraction, standard curve, and antiserum type. Please refer to the enclosed datasheet to determine these parameters and proceed as required for each step of the protocol.
DAY 1
1 - Dilute the RIA buffer concentrate to 200 ml
2 - Sample extraction is recommended especially for serum samples. It may not be as important for some tissue culture samples. See "Suggested Protocol for Sample Extraction" below for details. The kit may still be used without extraction but this may cause unexpected results due to the possible binding between serum proteins and kit components.
If you purchased an Extraction-Free Kit you may use it for the measurement of human or rat serum or plasma (according to its designation) without performing an extraction.
Sample concentration. The concentration of the target molecule must be within the measuring range of the kit (in a region around the IC50). If you cannot estimate the concentration range of your sample you can prepare it at different concentrations such that one of the samples may be within the measuring range.
3 - Reconstitute the standard in 1ml RIA buffer and make serial dilutions covering the kit's range as stated in the enclosed datasheet.
Caution: please note carefully the amount of standard provided and the kit's range.
The tables on the next page are included to facilitate the serial dilution process.
If you purchased an Extraction-Free Kit you should use the included Standard Diluent - (8 ml of peptide-free stripped frozen human or rat serum) to make the dilutions.
Note: the standards and the samples should be in the same diluent. If extraction was done the diluent should be RIA buffer. Otherwise, for extraction-free kits use your own diluent if possible or use the included standard diluent for standards to match the serum samples.
4 - Reconstitute the antiserum in 13 ml RIA buffer.
5- Mix 100 ml standards or samples with 100 ml antiserum.
Add no standard or sample to controls TB, NSB, and TB.
Add only 100 ml diluent to TB.
Add 100 ml diluent plus 100 ml RIA buffer to TC and NSB.
6 - Vortex and incubate overnight (16-24 hrs.) at 4°C
DAY 2
7- Tracer reconstitution and adjusting cpm
Reconstitute the 125I-peptide with 1 ml of RIA buffer and mix thoroughly (this is your stock solution). From this stock solution make a working tracer solution in RIA buffer at 10,000 to 15,000 cpm / 100 ml. The kit will be most sensitive with the least amount of tracer that will still provide accurate counts at the lower end of the assay range. We have determined empirically that 10,000 to 15,000 cpm per assay tube (TC cpm) works well. If you need help in making the correct dilution of tracer please do the following:
N = number of assays and standards (plus ~ 10%)
V = N x 0.1 ml (volume of needed RIA buffer in ml)
To = N x 2.5 ml (initial volume of tracer stock in ml)
Mix V ml RIA buffer with To ml tracer stock and count 100 ml
C100 = cpm in 100 ml initial dilution
Tadd= additional ml tracer that must be added to reach the maximum recommended 15,000 cpm/100ml
If you require a higher specific activity than 15,000 cpm / 100 µl simply plug in the desired number instead of 15,000. If you require less, plug in the new number and also start with a lower To
The unused tracer stock solution can be stored at 4oC for no more than 1 day to avoid freezing and thawing. If storing for a longer period of time, create aliquots of the tracer to prevent multiple freezing and thawing and store at -20oC or lower for maximum stability.
8- Add 100 ml diluted 125I tracer (e.g. ~10 Kcpm) to all tubes.
9- Vortex and incubate overnight (16-24 hrs.) at 4°C
DAY 3
10, 11 - Add secondary Ab and normal serum
Most of our RIA kits include goat-anti-rabbit-IgG (GARGG) as a secondary Ab and normal rabbit serum (NRS) but a few include goat-anti-guinea-pig-IgG (GAGGG) secondary Ab and guinea pig serum (NGS).
a) Reconstitute the secondary antibody (GARGG or GAGGG) with 13 ml of RIA Buffer.
b) Reconstitute the normal serum (NRS or NGS) with 13 ml of RIA Buffer.
c) Add 100 ml of secondary antibody and 100 ml of normal serum to each tube and vortex.
12 - Incubate at room temperature for 90 minutes
13 - Add 500 µl of RIA Buffer to each tube and vortex.
14 - Centrifuge for 20 minutes at 1700 x g.
15 - Set aside the TC tubes. DO NOT ASPIRATE THE TC TUBES. Aspirate all other tubes being careful not to disturb the pellet.
16 - Count, plot and analyze the cpm data.
Use a calibrated gamma counter and collect the cpm data for all tubes.
Should you need help for the plotting and analysis of your data we recommend the following procedure:
Set up a spreadsheet as shown below (note that the values on the spreadsheet are merely illustrative and are not necessarily typical for this particular kit). Please e-mail us at and we will be happy to send you the actual working Excel spreadsheet shown below.
For tubes labeled TC, NSB, TB, H, G, F, ..., A, enter the cpm from the duplicate tubes and average the values
Calculate all B/Bo values with the following formula:
Plot a standard curve y = f(x) on a semi-log scale where y is B/Bo and x are the standard concentrations in ng/ml.
We recommend that you use a good automatic data fitting computer program, but you may also obtain excellent results by fitting a four-parameter curve to the standard readings on a Excel sheet as shown here. Use the following equation to calculate the values on the "FIT" column. Then change the parameters a, b, c, and d, until you are satisfied that fit is good.